A major challenge in cancer chemotherapy is the selective delivery of small molecule anti-cancer agents to cancer cells. Doxorubicin (DOX) is a potent antineoplastic agent that is effective against a wide range of solid tumors and lymphomas but it is also associated with an irreversible cardiomyopathy above cumulative doses of 550 mg/m2 (Chabner B A, et al., Cytotoxic agents. In: Goodman and Gilman's the pharmacological basis of therapeutics. 12 ed. New York: McGraw-Hill, 2011). This and other toxic side effects make the drug a good candidate for localized drug delivery. DOX has been investigated in several macro-molecular delivery systems such as liposomes (Gabizon A, et al., Clin Pharmacokinet. 2003, 42, 419-36), synthetic copolymers of N-(2-hydroxypropyl)methacrylamide (HPMA) (Minko T, et al., Int J Cancer. 2000, 86, 108-17; Etrych T, et al., Macromol Biosci. 2002, 2, 43-52), other synthetic water soluble polymers (Duncan R, Vicent M J., Adv Drug Deliv. Rev. 2010, 62, 272-82), micelles (Matsumura Y, et al. Br J Cancer. 2004, 91, 1775-81; Kataoka K, et al. J Control Release Soc. 2000, 64, 143-53), polysaccharides (Lu D, et al., J Biomed Mater Res Part B: Appl Biomater. 2009, 89, 177-83) as well as block copolymer vesicles (or polymersomes) (Ghoroghchian P P, et al., Macromolecules. 2006, 39, 1673-5; Upadhyay K K, et al., Biomaterials. 2010, 31, 2882-92). Such delivery systems have demonstrated preferential accumulation in solid tumors compared to healthy tissue due to the enhanced permeation and retention effect (EPR) (Minko T, et al., Int J Cancer. 2000, 86, 108-17; Maeda H, J. Control Release Soc. 2000, 65, 271-84.). The resulting therapeutic advantages include an enhanced antitumor effect and reduced systemic toxicities (Minko T, et al., Int J Cancer. 2000, 86, 108-17; Duncan R., Nat Rev Drug Discov. 2003, 2, 347-60; Etrych T, et al., J Control Release Soc. 2008, 132, 184-92; Ayen W Y, Kumar N., Pharm Res. 2012, 29, 2522-33). Also, maximum tolerated doses of 5 to 10 fold greater than the free drug have been reported (Duncan R., Nat Rev Drug Discov. 2003, 2, 347-60; Sirova M, et al., Pharm Res. 2010, 27, 200-8). In addition, the ability to overcome drug resistance has been reported (Minko T, et al., J Control Release Soc. 1999, 59, 133-48; Nan A, et al., J Drug Target. 2005, 13, 189-97). These and similar delivery systems, however, have had concerns. An early HPMA-DOX conjugate showed little, if any, improved efficacy in Phase I clinical trials compared to the free drug (Vasey P A, et al. J Am Assoc Cancer Res. 1999, 5, 83-94). Mucocutaneous toxicities were reported from liposomal delivery of DOX (Ranson M R, et al., J Am Soc Clin Oncol. 1997, 15, 3185-91). And in a novel biodegradable delivery system not containing DOX, a polyglutamic acid carrier used with paclitaxol failed to demonstrate improved overall survival in Phase III clinical trials (Wang X, et al., Cancer Chemother Pharmacol. 2010, 65, 515-26).
Gelatin is the denatured and partially hydrolyzed product of collagen (Veis A. The macromolecular chemistry of gelatin. New York: Academic, 1964). It has been used as a macromolecular carrier to deliver several drugs including amphotericin B (Nahar M, et al., J Drug Target. 2010, 18, 93-105), methotrexate (Bowman B J, Ofner C M. Pharm Res. 2000, 17, 1309-15), and tumor necrosis factor (Tabata Y, et al., J Pharm Pharmacol. 1993, 45, 303-8). It has also been shown to have cell uptake (Ofner C M, et al., Int J Pharm. 2006, 308, 90-9). Its high molecular weight and biodegradability are attractive properties for use as a carrier in a DOX macromolecular delivery system. A sufficiently high molecular weight (e.g., 40 kDa or higher) can avoid glomerular filtration by the kidney leading to an extended circulation time and greater tumor accumulation by the EPR effect. Once the gelatin conjugate accumulates within the interstitial space of a tumor, its susceptibility to degradation by metalloproteinases, such as cathepsin B (Ofner C M, et al., Int J Pharm. 2006, 308, 90-9), would reduce the conjugate size and potentially enhance endocytotic uptake into the tumor cells. Recent reports describe encouraging results of high molecular weight HPMA-DOX conjugates containing cleavable links to allow breakdown in the body to lower molecular weight species (Etrych T, et al., J Control Release Soc. 2011, 154, 241-8; Etrych T, et al., J Control Release Soc. 2012, 164, 346-54). These lower sizes, however, are substantially larger than could occur with a biodegradable gelatin carrier.
Despite the interest in the art in synthesizing a high molecular weight gelatin-DOX conjugate, there are numerous synthetic challenges, particularly related to the degradation of high molecular weight gelatin during synthesis. For example, in an attempt to synthesize a high molecular weight gelatin-DOX conjugate (Wu et al., Pharm. Res. 2013, 20, 2087-2096), Wu et al. started with high molecular weight gelatin, but only produced low molecular weight gelatin-DOX conjugates (about 22 kDa). Accordingly, there is an unmet need in the art for high molecular weight gelatin-DOX conjugates and methods of production thereof.